One of the first events during the replication of HIV-1 and all other known retroviruses is conversion of genomic RNA to DNA using the viral reverse transcriptase (RT) and other host cell components (for review see Coffin, 1982; Varmus and Brown, 1989; Wain-Hobson, 1994). The viral RNA is reverse transcribed by RT with a host tRNA being used as the primer (reviewed in Wong-Staal, 1990; Vaishnav and Wong-Staal, 1991). Nucleocapsid protein plays an instrumental role in annealing of the primer to the genomic RNA. It has been suggested that nucleocapsid from Mason-Pfizer monkey virus (MPMV) and HIV-1 stimulates annealing of tRNA primer to viral RNA by over two orders of magnitude (Dib-Hajj et al., 1993). Moreover, it has been shown that the nucleocapsid proteins of Rous sarcoma virus (RSV) and murine leukemia virus(MuLV) are directly involved in annealing the tRNA primer onto the primer binding site under physiological conditions (Prats et al., 1988; Barat et al., 1989). In addition, nucleocapsid has been implicated in dimer formation of genomic RNA (Kung et al., 1976; Meric and Spahr, 1986; Prats et al., 1988; Bieth et al., 1990), non-specific coating of the viral RNA genome (Fleissnerand Tress, 1973; Chen et al., 1980), encapsidation of full-length genomic RNA (South and Summers, 1993) and interaction with RT during reverse transcription suggesting it may play a role in template transfer (Panganiban and Fiore, 1988; Barat et al. 1989).
Retroviral nucleocapsid proteins are gag precursor products (Mervis et al., 1988; Morellet et al., 1992). HIV-1 nucleocapsid protein contains two zinc finger binding domains with the following general structure Cys-X.sub.2 -Cys-X.sub.4 -His-X.sub.4 -Cys (SEQ ID NO: 26) (Gorelick et al., 1993). Mutations in each of these finger domains result in different functional and structural defects (Gorelick et al., 1990; Gorelick et. al., 1993; Julian et al., 1993). Similar types of mutations in RSV and MuLV nucleocapsid-proteins resulted in defects in RNA packaging and dimer formation (Meric and Spahr, 1986; Meric and Goff, 1989). Results from these studies suggested a role for nucleocapsid during reverse transcription (Meric and Goff, 1989; Weiss et al., 1992) and possibly during infection (Meric and Spahr, 1986). Other mutational studies demonstrated that substitutions of the conserved Cys residues in the Zn++ finger domain perturb sequence specific binding by nucleocapsid (Delahunty et al., 1992).
Sequences near the 5' end of the mature HIV-1 viral RNA are involved in encapsidation (Aldovini and Young, 1990). It appears these sequences interact specifically with the nucleic acid binding zinc finger domains of HIV-1 nucleocapsid (NC). Mutations in either of the two Zn++ fingers or the encapsidation site resulted in similar defects. The 5' end of retroviral RNAs have been rigorously studied and thus the structural and functional characterization is extensive (Bender et al., 1978; Murti et al., 1981; Darlix et al., 1982; Aldovini and Young, 1990; Harrison and Lever, 1992; Surovoy et al., 1993).
A method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules has been developed. This method, Systematic Evolution of Ligands by EXponential enrichment, termed SELEX, is described in U.S. patent application Ser. No. 07/536,428, entitled "Systematic Evolution of Ligands by Exponential Enrichment," now abandoned, U.S. patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled "Nucleic Acid Ligands now issued as U.S. Pat. No. 5,475,096," U.S. patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled "Nucleic Acid Ligands," now U.S. Pat. No. 5,270,163 (see also PCT/US91/04078), each of which is herein specifically incorporated by reference. Each of these applications, collectively referred to herein as the SELEX Patent Applications, describes a fundamentally novel method for making a nucleic acid ligand to any desired target molecule.
The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific, high affinity nucleic acid ligands to the target molecule.
The basic SELEX method has been modified to achieve a number of specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, entitled "Method for Selecting Nucleic Acids on the Basis of Structure" now abandoned, describes the use of SELEX in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled "Photoselection of Nucleic Acid Ligands" now abandoned describes a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993, entitled "High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine," now abandoned describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, termed Counter-SELEX. U.S. patent application Ser. No. 08/143,564, filed Oct. 25, 1993, entitled "Systematic Evolution of Ligands by EXponential Enrichment: Solution SELEX," now abandoned describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low. affinity for a target molecule. U.S. patent application Ser. No. 07/964,624, filed Oct. 21, 1992, entitled Nucleic Acid Ligands to HIV-RT and HIV-1 Rev now issued as U.S. Pat. No. 5,496,938 describes methods for obtaining improved nucleic acid ligands after SELEX has been performed. U.S. patent application Ser. No. 08/400,440, filed Mar. 8, 1995, entitled "Systematic Evolution of Ligands by EXponential Enrichment: Chemi-SELEX," describes methods for covalently linking a ligand to its target.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX-identified nucleic acid ligands containing modified nucleotides are described in U.S. patent application Ser. No. 08/117,991, filed Sep. 8, 1993, entitled "High Affinity Nucleic Acid Ligands Containing Modified Nucleotides" now abandoned, that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2'-positions of pyrimidines. U.S. patent application Ser. No. 08/134,028, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2'-amino (2'-NH.sub.2), 2'-fluoro (2'-F), and/or 2'-O-methyl (2'-OMe). U.S. patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled "Novel Method of Preparation of known and novel 2' Modified nucleosides by Intramolecular Nucleophilic Displacement," describes oligonucleotides containing various 2'-modified pyrimidines.
The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. patent application Ser. No. 08/284,063, filed Aug. 2, 1994, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEX" and U.S. patent application Ser. No. 08/234,997, filed Apr. 28, 1994, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Blended SELEX," respectively. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules. Each of the above described patent applications which describe modifications of the basic SELEX procedure are specifically incorporated by reference herein in their entirety.
Parent U.S. patent application Ser. No. 08/447,172 (Express Mail Receipt No. TB865017604US), entitled High Affinity HIV-1 GAG Nucleic Acid Ligands, filed May 19, 1995, which is hereby incorporated by reference in its entirety, describes a method for identifying nucleic acid ligands to HIV-1 GAG polyprotein, and the nucleic acids so produced. Nucleocapsid is a component of the GAG polyprotein.
HIV-1 nucleocapsid is an attractive target for the development of SELEX ligands because HIV-1 nucleocapsid plays such a crucial role in so many different viral processes.
The development of high affinity ligands capable of inhibiting HIV-1 nucleocapsid would be useful in the treatment of Human Immunodeficiency Virus. Herein described are high affinity RNA ligand inhibitors of HIV-1 nucleocapsid.